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Nucleic Acids Symposium Series 2009 53(1):37-38; doi:10.1093/nass/nrp019
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© 2009 Oxford University Press

This article appears in the following Nucleic Acid Symposium Series issue: The 6th International Symposium on Nucleic Acids Chemistry (36th Symposium on Nucleic Acids Chemistry) [View the issue table of contents]

The NEXT-A (N-Terminal EXtension with Transferase and ARS) reaction

Masumi Taki1,*, Hiroyuki Kuroiwa1 and Masahiko Sisido1,*

1The Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-0082, Japan

*Corresponding authors. E-mail: taki{at}biotech.okayama-u.ac.jp sisido{at}cc.okayama-u.ac.jp

Abstract

L/F-transferase is known to catalyze transfer of hydrophobic amino acids from aminoacyl tRNA to the N-terminus of a protein possessing lysine or arginine as the N-terminus. Combining L/F-transferase with E. coli phenylalanyl-tRNA synthetase (ARS), we achieved non-ribosomal N-terminal-specific introduction of various kinds of nonnatural amino acids to a protein. A nonnatural amino acid is once charged onto an E. coli tRNAPhe by a mutant ARS in situ, and successively transferred from the tRNA to a target protein, namely the NEXT-A reaction. Besides {alpha}A294G mutation on the ARS, {alpha}T251A, βG318W, or βA356W double-mutation were effective to increase the introduction efficiency through the NEXT-A reaction. Protein specific fluorescence labelling via the NEXT-A reaction followed by Huisgen cycloaddition was also demonstrated.


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